EP3805255A1 - Protéine immunogène d'acinetobacter baumannii, composition et utilisation correspondantes - Google Patents
Protéine immunogène d'acinetobacter baumannii, composition et utilisation correspondantes Download PDFInfo
- Publication number
- EP3805255A1 EP3805255A1 EP19806420.6A EP19806420A EP3805255A1 EP 3805255 A1 EP3805255 A1 EP 3805255A1 EP 19806420 A EP19806420 A EP 19806420A EP 3805255 A1 EP3805255 A1 EP 3805255A1
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- Prior art keywords
- protein
- acinetobacter baumannii
- ata
- fusion protein
- nucleic acid
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
- C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
- A61K39/1045—Moraxella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1218—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Acinetobacter
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55544—Bacterial toxins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
Definitions
- the invention relates to the field of biotechnology, especially relates to Acinetobacter baumannii immunogenic protein and its composition and application, in particular, it relates to an immunogenic fusion protein comprising a component of Acinetobacter baumannii Ata protein, a vaccine composition containing the protein, and application thereof in a method for immunizing animals or humans to resist Acinetobacter baumannii infection.
- Acinetobacter baumannii is a gram-negative bacterium that exists widely in nature. It is a kind of opportunistic pathogenic bacteria. It is highly susceptible to infections in people with weakened immune functions. It is also one of the most typical clinical pathogens in hospital infections. Because of its fast proliferation and strong adhesion, it is widely distributed in the hospital environment, and with the large-scale use of antibiotics, more and more drug-resistant strains have appeared. Acinetobacter baumannii has posed a serious threat to the global healthcare system. At present, antibiotics are mainly used clinically to combat Acinetobacter baumannii infection. However, because of the emergence of multi-drug resistant strains, the doctors have to combine medication during treatment. But there are many disadvantages in combination medication. Therefore, the development of related vaccines is urgent for preventing the epidemic of Acinetobacter baumannii. Unfortunately, there is no vaccine against Acinetobacter baumannii on the market.
- the present invention aims to provide an immunogenic fusion protein containing a fragment of Acinetobacter baumannii Ata protein, a vaccine composition containing the protein and its application in a method for immunizing mammals against Acinetobacter baumannii infection.
- the inventors of the present invention found that the fusion protein, formed by component of the surface protein Ata (Acinetobacter trimeric autotransporter) of Acinetobacter baumannii fused with the adjuvant protein, has immunogenicity that meets the requirements of vaccines for Acinetobacter baumannii and can be used to prepare the vaccine for preventing Acinetobacter baumannii infection and apply the vaccine. Based on the above findings, the inventors completed the present invention.
- an immunogenic fusion protein comprising a component of the Ata protein of Acinetobacter baumannii, the component comprising the amino acid sequence fragment of the N-terminal ⁇ -helix part of the Ata protein transport functional domain of Acinetobacter baumannii; and adjuvant protein or protein fragment with adjuvant function.
- Another aspect of the present invention provides a nucleic acid molecule encoding the aforementioned fusion protein.
- biomaterial selected from:
- Another aspect of the present invention provides a method for protecting animals or humans from infection of Acinetobacter baumannii, comprising the following steps: immunize animals or humans in need with aforementioned protein composition or the aforementioned fusion protein or the aforementioned nucleic acid molecule or the aforementioned biomaterial to achieve resistance to Acinetobacter baumannii infection.
- Another aspect of the present invention provides a method for producing Acinetobacter baumannii antibodies, comprising the following steps: effectively immunize animals or humans with aforementioned protein composition or the aforementioned fusion protein or the aforementioned nucleic acid molecule or the aforementioned biomaterial to obtain Acinetobacter baumannii antibodies.
- Aforementioned immunization may be intraperitoneal immunization or subcutaneous immunization.
- Another aspect of the present invention provides a product comprising the aforementioned protein or the aforementioned protein composition or the aforementioned fusion protein or the aforementioned nucleic acid molecule or the aforementioned biomaterial or the aforementioned antibodies.
- Another aspect of the present invention provides the use of the fusion protein of the present invention or its encoding nucleic acid molecule in preparing a vaccine composition against Acinetobacter baumannii infection.
- protein used in the present invention refers to molecular chains of amino acids, including peptides, oligopeptides and polypeptides. If necessary, the said protein can be modified in vivo or in vitro by, for example, glycosylation, amidation, carboxylation or phosphorylation.
- the protein or peptide can be natural or synthetic.
- fusion protein as used in the present invention means a protein formed by two or more polypeptides that are directly or indirectly covalently linked to each other in a certain manner.
- immunoprotecting means the response ability (partially or fully) of serum antibodies and/or cytotoxic T cell induced during immunization, which prevents diseases caused by such as Acinetobacter baumannii.
- the term "homology" used in the present invention refers to the homology of DNA nucleotide and protein amino acid sequences.
- the former can be determined by DNA sequencing methods, and the latter can be determined by methods such as mass spectrometry or Edman degradation.
- an immunogenic protein composition comprising 39 amino acids of the N-terminal ⁇ -helix part of the Ata protein transport functional domain of Acinetobacter baumannii and an adjuvant protein or protein fragment with adjuvant function.
- an immunogenic fusion protein which comprises a component of Acinetobacter baumannii Ata protein, this component contains the 39 amino acids of the N-terminal ⁇ -helix part of the Ata protein transport functional domain of Acinetobacter baumannii, that is, the 127 th -165 th amino acid residues in SEQ ID NO:2; and an adjuvant protein or protein fragment with adjuvant function.
- the adjuvant protein is specifically the cholera toxin B subunit, namely CTB protein.
- the aforementioned fusion protein is any protein of the following a)-e):
- nucleic acid molecule encoding the aforementioned fusion protein, and the nucleic acid molecule is a nucleic acid molecule shown in any of the following 1)-4):
- biomaterial selected from:
- a method for making animals or humans resistant to infection by Acinetobacter baumannii which comprises the following steps: immunize animals or humans with the protein or protein composition or fusion protein or nucleic acid molecule or biomaterial of any of the aforementioned schemes to achieve resistance to Acinetobacter baumannii infection.
- a method for producing Acinetobacter baumannii antibodies which comprises the following steps: immunize animals or humans with the protein or protein composition or fusion protein or nucleic acid molecule or biomaterial of any of the aforementioned schemes to obtain the Acinetobacter baumannii antibodies.
- a product which comprises the protein or protein composition or fusion protein or nucleic acid molecule or biomaterial or antibodies of any of the aforementioned embodiments.
- the product of the present invention has at least one of the following a-c functions: a) Promote animals or humans to produce antibodies against Acinetobacter baumannii; b) Prevent and/or treat diseases caused by Acinetobacter baumannii ; c) Resist infestation or infection by Acinetobacter baumannii.
- said product is medicine or health product or detection kit or vaccine.
- said animal is mouse.
- Example 1 Obtaining the fusion protein CTB-Ata
- Fusion protein CTB-Ata comprises CTB amino acid sequence and 39 amino acids of the N-terminal ⁇ -helix part of the surface protein Ata ( Acinetobacter trimeric autotransporter) transport functional domain of Acinetobacter baumannii.
- the 39 amino acids of the ⁇ -helix part of the Ata protein, the tac promoter, and the CTB protein for fusion expression were synthesized by whole gene to form the fusion protein CTB-Ata.
- the amino acid sequence of the fusion protein is shown as SEQ ID NO:2 in the sequence listing.
- the 20 th -123 th amino acids residues in SEQ ID NO:2 represent the fusion expressed CTB protein
- the 127 th -165 th amino acids residues represent the ⁇ -helix part of Ata protein
- the 201 th -206 th amino acids residues represent the His tag.
- the nucleotide sequence of the fusion protein encoding gene CTB-Ata is shown as SEQ ID NO:1 in the sequence listing, in which nt103-131 is the tac promoter, nt235-543 is the coding gene for CTB protein, and nt556-672 is the coding gene for alpha-helix part of Ata protein, and nt778-795 is the His tag gene.
- the recombinant expression vector pET28a-CTB-Ata is a vector obtained by replacing the DNA molecule between the XbaI and XhoI restriction sites of the pET28a(+) vector with the fusion protein encoding gene CTB-Ata shown in SEQ ID NO: 1. This fusion protein contains His tag of the vector.
- the recombinant expression vector pET28a-CTB-Ata obtained in aforementioned 2 was introduced into BL21 cells to obtain recombinant strain pET28a-CTB-Ata/BL21.
- the recombinant strain pET28a-CTB-Ata/BL21 was inoculated into 5ml LB liquid medium containing a final concentration of 50 ⁇ g/mL kanamycin, cultivated overnight at 37°C, and passaged in LB liquid medium at a volume ratio of 1:100, cultivated at 37°C until OD 600 was about 0.6, added IPTG with a final concentration of 1 mM, reduced the temperature to 30°C and induced for 12 hours, obtained protein induction culture solution, centrifuged (10800g for 8 minutes), collected the precipitate to obtain protein induction bacteria.
- the aforementioned supernatant was purified using a Chelating affinity chromatography column (GE Healthcare, product catalog number 17-5203-06) ( ⁇ 1.6cm ⁇ 15cm). First flushed the column bed with 0.5M NaOH aqueous solution for at least 3 column beds volume, then balanced with deionized water to neutral pH, then used 0.5M NiSO 4 aqueous solution to equilibrate at least 3 column beds volume.
- the purified fusion protein CTB-Ata was analyzed by 15% SDS-PAGE and detected by WB with anti-His antibodies. The result is shown in Figure 2 . After purification, a relatively pure target fusion protein CTB-Ata (molecular weight 22.5 kDa) was successfully obtained. After protein quantification by BCA method, the following animal experiments were carried out.
- the empty vector pET28a was transferred into BL21 cells, and the expression was induced in the same way, but the target protein was not obtained.
- Example 2 Diluted the purified fusion protein CTB-Ata prepared in Example 1 with physiological saline to prepare an immune sample containing the fusion protein CTB-Ata.
- the immunization was carried out according to the immunization dose of 2.5 ⁇ g of fusion protein CTB-Ata per mouse (mouse weight was about 17g), and immunization with the Ata peptide fragment (39 amino acids of N-terminal ⁇ -helix part) was as a control.
- mice Took 50 5-week-old female Balb/c mice (Beijing Vital River Laboratory Animal Technology Co. Ltd) (with no significant difference in body weight) and randomly divided them into 5 groups (10 mice in each group) :
- Blank control group (no sample injected, control): female Balb / c mice.
- Each group was immunized on the 1st, 14th, and 28th day. After each immunization injection, cut the tail and collected blood, and collected the serum of each group of mice.
- the indirect ELISA method was used to detect the titer of antibody against Acinetobacter baumannii in the serum of each group of mice, details as follows:
- each mouse in each group (10 mice) was challenged by intraperitoneal injection with 1.5 times the LD 50 of Acinetobacter baumannii. Each mouse was injected with a volume of 200 ⁇ l. Observed and recorded the number of dead mice in each group for 7 consecutive days.
- the 39 amino acids of the N-terminal ⁇ -helix part of surface protein Ata (Acinetobacter trimeric autotransporter) of Acinetobacter baumannii were taken and fused with CTB, and expressed in BL21. Purifying by a nickel column, and immunizing with 2.5 ⁇ g/mouse intraperitoneally or subcutaneously, which verified its immunogenicity and immunoprotection through the animal experiments, and proved that it has a good effect of anti-infection of Acinetobacter baumannii.
- the protein was purified by a nickel column, and used to intraperitoneally or subcutaneously immunize mouses by 2.5 ⁇ g/mouse. Its immunogenicity and immunoprotection was verified through the animal experiments, which proves it has a good effect of anti-infection of Acinetobacter baumannii..
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- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
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- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
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- Public Health (AREA)
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- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
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- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810510029.2A CN108503698B (zh) | 2018-05-24 | 2018-05-24 | 鲍曼不动杆菌Ata蛋白的免疫原性 |
PCT/CN2019/088074 WO2019223749A1 (fr) | 2018-05-24 | 2019-05-23 | Protéine immunogène d'acinetobacter baumannii, composition et utilisation correspondantes |
Publications (2)
Publication Number | Publication Date |
---|---|
EP3805255A1 true EP3805255A1 (fr) | 2021-04-14 |
EP3805255A4 EP3805255A4 (fr) | 2022-05-04 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP19806420.6A Pending EP3805255A4 (fr) | 2018-05-24 | 2019-05-23 | Protéine immunogène d'acinetobacter baumannii, composition et utilisation correspondantes |
Country Status (4)
Country | Link |
---|---|
US (1) | US20210214400A1 (fr) |
EP (1) | EP3805255A4 (fr) |
CN (1) | CN108503698B (fr) |
WO (1) | WO2019223749A1 (fr) |
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CN108503698B (zh) * | 2018-05-24 | 2021-03-05 | 中国人民解放军军事科学院军事医学研究院 | 鲍曼不动杆菌Ata蛋白的免疫原性 |
CN110724201B (zh) * | 2019-10-31 | 2023-01-24 | 湖北工业大学 | 基于多重表位融合抗原的鲍曼不动杆菌感染快速检测法 |
CN110713524B (zh) * | 2019-10-31 | 2023-02-03 | 湖北工业大学 | 一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒 |
CN110713523B (zh) * | 2019-10-31 | 2023-02-03 | 湖北工业大学 | 一种定性检测人血清中鲍曼不动杆菌特异性抗体的检测条 |
CN110950939B (zh) * | 2019-12-04 | 2022-05-17 | 南京医科大学第二附属医院 | 鲍曼不动杆菌Omp22重组多抗原表位多肽及其应用 |
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WO2017004545A1 (fr) * | 2015-07-01 | 2017-01-05 | Ketter Patrick | Procédés et compositions pour prévenir ou traiter des infections bactériennes |
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CN108503698B (zh) * | 2018-05-24 | 2021-03-05 | 中国人民解放军军事科学院军事医学研究院 | 鲍曼不动杆菌Ata蛋白的免疫原性 |
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2018
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2019
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WO2019223749A1 (fr) | 2019-11-28 |
CN108503698B (zh) | 2021-03-05 |
US20210214400A1 (en) | 2021-07-15 |
CN108503698A (zh) | 2018-09-07 |
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